ROS Promote Ox-LDL-Induced Platelet Activation by Up-Regulating Autophagy Through the Inhibition of the PI3K/AKT/mTOR Pathway
ROS Promote Ox-LDL-Induced Platelet Activation by Up-Regulating Autophagy Through the Inhibition of the PI3K/AKT/mTOR Pathway
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ROS 通过抑制 PI3K/AKT/mTOR 通路上调自噬，促进 Ox-LDL 诱导的血小板活化
DOI：
10.1159/000494795
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发表时间：
2018-01-01
影响因子：
--
通讯作者：
Chen, Fang-Ping
中科院分区：
文献类型：
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作者：
Wang, Xiang;Fu, Yun-Feng;Chen, Fang-Ping
背景/目的：氧化型低密度脂蛋白(OxLDL)可促进血脂紊乱患者的无调节的血小板活化。尽管oxLDL刺激激活信号，但研究人员还没有清楚地确定这些事件是如何推动加速血栓形成的。在这里，我们描述了ROS通过调节PI3K/AKT/mTOR信号通路来调节氧化低密度脂蛋白诱导的血小板激活过程中的自噬的机制。方法：体外实验分别用氧化低密度脂蛋白、ROS清除剂N-乙酰半胱氨酸(NAC)、mTOR抑制剂雷帕霉素和自噬抑制剂3-MA单独或与其他化合物联合作用。然后在聚集仪上检测血小板聚集性，并在剪切力作用下测量血小板粘附性。用流式细胞仪检测血小板活化标志物CD62p的水平，用流式细胞仪测定DCFH-DA的荧光强度来定量测定ROS水平。分别用硝酸还原酶法和荧光素增强化学发光法测定一氧化氮(NO)和超氧化物歧化酶(O2·-)水平。用透射电子显微镜观察自噬小体的形成，免疫荧光染色检测Lc3的表达，Western blotting检测PI3K/AKT/mTOR通路和自噬相关蛋白的水平。结果：氧化低密度脂蛋白诱导的血小板聚集和黏附、CD62p表达、ROS水平和O2·-含量显著增加，Lc3II/Lc3I比值和Beclin1表达增加，而p62和通路相关蛋白水平显著降低(均P<0.05)。然而，雷帕霉素治疗可加重血小板活化和自噬作用，经NAC、3-MA或NAC和3-MA联合治疗后，血小板活化和自噬作用减弱，同时PI3K/AKT/mTOR通路的活性增加。此外，与单用雷帕霉素组相比，NAC+雷帕霉素或雷帕霉素+3-MA组的血小板活化和自噬能力降低，提示NAC和3-MA均能逆转雷帕霉素的作用。结论：抑制ROS的产生可能通过激活PI3K/AKT/mTOR通路减少自噬，从而抑制ox-LDL诱导的血小板活化。
Background/Aims: Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in patients with dyslipidemic disorders. Although oxLDL stimulates activating signaling, researchers have not clearly determined how these events drive accelerated thrombosis. Here, we describe the mechanism by which ROS regulate autophagy during ox-LDL-induced platelet activation by modulating the PI3K/AKT/mTOR signaling pathway. Methods: For in vitro experiments, ox-LDL, the ROS scavenger N-acetylcysteine (NAC), the mTOR inhibitor rapamycin and the autophagy inhibitor 3-MA were used alone or in combination with other compounds to treat platelets. Then, platelet aggregation was evaluated on an aggregometer and platelet adhesion was measured under shear stress. The levels of a platelet activation marker (CD62p) were measured by flow cytometry, reactive oxygen species (ROS) levels were then quantified by measuring DCFH-DA fluorescence intensity via flow cytometry. Nitric oxide (NO) and superoxide (O2·-) levels were determined by the nitric acid deoxidize enzyme method and lucigenin-enhanced chemiluminescence (CL), respectively. Transmission electron microscopy was used to observe the autophagosome formation, immunofluorescence staining was employed to detect LC3 expression and western blotting was used to measure the levels of PI3K/AKT/mTOR pathway- and autophagy-related proteins. Results: Ox-LDL-induced platelets showed a significant increase in platelet aggregation and adhesion, CD62p expression, ROS level and O2·- content, with an elevated LC3II/LC3I ratio and Beclin1 expression, but a dramatic reduction in the levels of p62 and pathway-related proteins (all P < 0.05). However, platelet activation and autophagy were aggravated by the Rapamycin treatment, and decreased following treatment with NAC, 3-MA, or NAC and 3-MA, together with increased activity of the PI3K/AKT/mTOR pathway. Additionally, decreased platelet activation and autophagy were observed in platelets treated with NAC and Rapamycin or Rapamycin and 3-MA compared with platelets treated with Rapamycin alone, suggesting that both NAC and 3-MA reversed the effects of Rapamycin. Conclusion: Inhibition of ROS production may reduce autophagy to suppress ox-LDL-induced platelet activation by activating PI3K/AKT/mTOR pathway.